CN110308288B - Novel blood platelet cross matching kit - Google Patents
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Abstract
The invention discloses a novel platelet cross matching kit, which comprises: a U-shaped reaction microplate, a low-ion medium, an indication system, negative control serum and positive control serum. Wherein, the inner surface of the U-shaped reaction microplate is coated with polyethyleneimine, and platelets are adsorbed to the bottom of the microplate by utilizing the high adsorbability and the high cationic flocculation of the polyethyleneimine; the indicator system comprised IgG anti-D sensitized O-type erythrocytes and rabbit or sheep anti-human globulin reagent. Adding plasma to be matched into the microplate hole with the adsorbed platelet monolayer for immunoreaction, and finally displaying by a two-step method indicating system to quickly realize the cross matching of the platelets by a solid phase method. The invention truly solves the problems of low sensitivity, high price of the kit and the like in the current clinical platelet cross matching method by innovatively utilizing the high flocculation adsorption effect of polyethyleneimine and utilizing a high-sensitivity two-step method indication system, and provides a novel kit with high cost performance for clinical platelet cross matching.
Description
Technical Field
The invention relates to a kit, belongs to the technical field of biology, and particularly relates to a novel platelet cross matching kit.
Background
Platelet infusion is widely used clinically as an effective treatment means. However, due to the influence of long-term blood transfusion, drug therapy and diseases, alloantibodies against platelet antigens are easily generated in the body of a patient, and after the antibodies act on human platelets, the antibodies can cause the ineffective platelet transfusion, purpura after blood transfusion and the like, so that the life safety of the patient is endangered. In view of this, the national health council stipulates that cross-matching experiments must be performed before platelet infusion to improve the blood transfusion quality of patients and ensure the life safety of patients.
The method for cross matching of the platelets sequentially goes through stages of a platelet immunofluorescence experiment (PIFT), flow cytometer cross matching (FCM), microcolumn gel matching, solid phase agglutination method (SPRCA) matching and the like. The PIFT method is mainly characterized in that platelets of a blood donor and serum of a patient are incubated, then the blood donor and the serum are incubated with anti-human globulin labeled with fluorescein isothiocyanate, and the blood donor and the serum are observed under a fluorescence microscope after being washed. The method can avoid non-specific reaction caused by some cell debris, and has the defect of insensitive reaction. The FCM technology is that after blood platelet of donor and serum of patient are incubated, fluorescein labeled antihuman-IgG is added, after light-shielding reaction, PBS is added for suspension, and detection and analysis are carried out on a computer. The method is sensitive, and has the defects of long experimental time, need of expensive instruments and equipment and fluorescent labeled antibodies, and difficulty in popularization and application in medical institutions. The micro-column gel card method for the cross matching experiment of the platelet is a new technology based on the traditional platelet detection and the immune micro-column gel. Platelets, serum and indicator red blood cells were added to the reaction chamber of the microcolumn, and the results were observed after incubation and centrifugation. The method is simple and quick to operate, does not need particularly expensive equipment, and has the defects of high false positive rate and certain difficulty in judging results. The SPRCA method is characterized in that platelets are added into a coated lath to form a platelet molecular monolayer by centrifugation, then patient serum is added, indicator red blood cells are added after incubation and washing, and the result is interpreted. The method has the advantages of good sensitivity, strong specificity, simplicity, convenience and rapidness, can be completed without special equipment, and is the mainstream technology in the field of platelet matching at present. Because most of products using the method adopt anti-platelet antibodies for coating at present, on one hand, the cost is relatively high; on the other hand, the coating matrix of the conventional solid phase agglutination method has a poor immobilization effect on the antigen substance and requires some special steps, thereby limiting its clinical application. Therefore, there is a need for an improvement of the conventional solid phase agglutination method.
Platelets, a specific blood component, contain multiple glycoproteins on their surface, and thus, the platelet surface is loaded with a large amount of negative charges. Under the action of the polymeric cation, negative charges loaded on the surface of the platelet can be neutralized to cause adsorptive aggregation of the platelet.
Polyethyleneimine is a water-soluble high-molecular polymer and has some special properties. 1. High adhesion and adsorption: polyethyleneimine can be combined with various substances due to its polar group (amino group) and hydrophobic group (vinyl group) structure. By utilizing the comprehensive binding force, the adhesive can be widely applied to the fields of adhesion, printing ink, coating, adhesive and the like. 2. High cationic flocculation property: the polyethyleneimine exists as a polymeric cation in water, can neutralize and adsorb all anionic substances, and can chelate heavy metal ions. The high cationic property of the cationic surfactant can be used in the fields of papermaking water treatment, electroplating solution and dispersing agent.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the platelet matching kit which has good sensitivity and moderate price, is not easy to cause false positive or false negative and can be used for clinical first-line detection.
The invention is to coat polyethyleneimine in a 96-well microplate by utilizing high adhesiveness thereof, adsorb platelets to the bottom of the microplate well by utilizing high adsorbability and high cationic flocculation thereof, after washing, add plasma to be detected and low ionic liquid for promoting reaction in the microplate well, so as to quickly realize immunological binding between platelet antigen and plasma antibody to be detected, and finally indicate by a high-sensitivity indicating system, wherein a positive reaction shows a uniform and consistent indicating erythrocyte monolayer on the bottom surface of the microplate well; negative reactions will present a rounded indicator red blood cell button on the bottom surface of the microplate well center. The invention has the innovation points that the polyethyleneimine is coated at the bottom of the pore of the microplate by utilizing the high adhesiveness of the polyethyleneimine, the high-strength adsorption is generated on the platelets by innovatively utilizing the adsorbability of the polyethyleneimine and the flocculation effect of the polymeric cation, and a high-sensitivity two-step method indicating system is utilized to replace a one-step method indicating system, so that the problems of low sensitivity, high price and the like in the conventional clinical platelet cross matching method can be practically solved, and the high-cost-performance platelet cross matching kit is provided for the first-line clinical detection work
A novel platelet cross-matching kit comprising:
(1) The device comprises a U-shaped solid-phase reaction microplate, wherein the bottom of a hole of the U-shaped solid-phase reaction microplate is coated with polyethyleneimine;
(2) A low ionic medium which is a buffer solution containing 1.8% of glycine and 0.10% of sodium azide;
(3) An indicator system comprising an IgG anti-D sensitized erythrocyte suspension and a rabbit or goat anti-human globulin reagent;
(4) Negative control serum without platelet antibody, wherein the negative control serum is prepared by adding 0.1% of sodium azide into human serum which has no agglutination reaction with human platelets;
(5) The positive control serum contains platelet antibodies, and the positive control serum is prepared by adding 0.1% of sodium azide into human serum which has agglutination reaction with human platelets.
In order to further realize the invention, the U-shaped reaction microplate is made of a 96-hole U-shaped bottom detachable microplate strip.
In order to further realize the invention, the indicator cells are prepared into 0.35% erythrocyte suspension after 0 type RhD (+) erythrocytes are sensitized by IgG-anti-D.
The invention has the beneficial effects that:
the U-shaped reaction microplate is made of polystyrene and does not participate in chemical reaction. The inner surface of the platelet is coated with 0.1% of polyethyleneimine, and the polyethyleneimine belongs to cationic polymers and can adsorb negative charges on the surface of the platelet. When the blood platelet contacts the polyethyleneimine at the bottom of the hole of the U-shaped reaction microplate, the blood platelet is adsorbed. Different from the conventional mode that platelet antibodies are coated at the bottom of a microplate and platelets are adsorbed by utilizing antigen-antibody reaction, the adsorption force of the bottom of a microplate hole on the platelets is greatly enhanced after the microplate is coated with polyethyleneimine, and the microplate is not limited by platelet antigens. Platelets diluted to an appropriate concentration (e.g., 100X 109/mL) are immobilized at the bottom of the microplate U-shaped well and can be used in the relevant experiments.
The invention provides a novel platelet cross matching kit, which is characterized in that a 96-hole U-shaped micro-lath is coated by polyethyleneimine in the aspect of coating a matrix, so that the adsorption capacity to platelets is strong, the platelet cross matching kit is not influenced by the specificity of platelet antigens, and the phenomenon that partial platelets cannot be paved is avoided; after the polyethyleneimine is coated on the bottom of the U-shaped hole, the stability is good, and the detection results of the kit in each sampling detection time period within 12 months are not different; meanwhile, the indication system designed by the invention adopts low-titer anti-D sensitized O-type RhD positive erythrocytes and is matched with a proper anti-human globulin reagent, so that the indication sensitivity is greatly improved. The matching method is simple and quick, does not need special equipment, can be used for single-person detection and large-scale detection, thereby reducing the detection cost, providing convenience for clinical detection and having better clinical application value.
Drawings
FIG. 1 is a graph showing the standard for the agglutination intensity of the solid phase agglutination assay kit;
FIG. 2 is a diagram of the use of the kit for cross-matching of platelets;
wherein, the kit micro-strips are marked as 1, 2, 3, 4, 5, 6, neg (negative control) and Pos (positive control) from top to bottom in sequence. Wherein 1-6 wells are added with platelets from different individuals and plasma of the same patient, and 3 human platelets of the 6 platelets are mixed and then added with Neg wells and Pos wells, and then respectively added with negative control plasma and positive control plasma. The results show that: the 1 st, 2 nd and 4 th hole platelets are negative in cross matching with the patient, the 3 rd and 5 th hole platelets are strong positive in matching (4 +) and weak positive in matching (2 +) with the 6 th hole platelets.
FIG. 3 is a graph of the stability test of the kit;
in this case, the kit stored in a refrigerator at 4 ℃ was cross-matched with platelets using a specimen (containing HLA antibody and HPA antibody) of the same patient every 2 months. The kit micro-strip is marked with 1, 2, 3, 4, 5, 6, neg (negative control) and Pos (positive control) from top to bottom in sequence. Wherein 1-6 wells are added with platelets from different individuals and plasma of the same patient, and 3 human platelets of the 6 platelets are mixed and then added with Neg wells and Pos wells, and then respectively added with negative control plasma and positive control plasma. The results show that: within 12 months of preservation, the kit has no difference on the platelet cross matching result of the positive patient, and the matching result of each stage is strong positive (4 +).
Detailed Description
The present invention is further explained below with reference to examples. It should be understood that the illustrated embodiments are only for explaining the present invention and are not limited to the protection scope of the present invention.
Example 1 preparation of platelet typing kit
Preparation of U-shaped reaction microplate
Preparing 0.1% polyethyleneimine solution by using 0.05M carbonate buffer solution with pH of 9.6, adding 50 uL/well into a hole of a 96-hole U-shaped (12 x 8) reaction plate, placing the reaction plate in a refrigerator at 4 ℃ overnight, taking out the reaction plate the next day, washing the reaction plate for 3 times by using 1 x PBS buffer solution, throwing off the washing solution, sucking residual liquid in the hole by using absorbent paper for the last time, bagging the reaction plate by using an aluminum foil bag, heating and sealing the reaction plate, and placing the reaction plate in the refrigerator at 4 ℃ for storage and standby.
Platelet harvesting
The blood platelet can be prepared by directly adopting machine-collected blood platelet or blood platelet-rich plasma. When the platelet rich plasma is adopted to prepare the platelets, a plurality of milliliters of freshly collected EDTA anticoagulated whole blood is taken, centrifuged at 1500rpm for 10 minutes, and then 2/3 of the platelet rich plasma is sucked. After counting the platelets, they were diluted to 100X 109/ml with PBS and ready for use.
Preparation of the indicator System
Preparation of sensitized erythrocytes: adding equal volume into washed O-shaped packed red blood cells
Incubating the IgG anti-D for 50 minutes at 37 ℃ to prepare IgG anti-D sensitized erythrocytes, washing the IgG anti-D sensitized erythrocytes for 5 times by using normal saline, and preparing 0.2-0.6% of suspension by using erythrocyte preservation solution, namely sensitized erythrocytes;
anti-human globulin reagent: rabbit or sheep anti-human globulin reagent carries out ladder with antibody diluent
Degree-fold dilution (e.g., 1:8, 1;
preparation of low ionic medium
Weighing 1.8g of glycine and 0.1g of sodium azide, putting the glycine and the sodium azide into a measuring cylinder, adding a small amount of pure water to dissolve, then adding the pure water to a constant volume of 100mL, and adjusting the pH value to 6.80 by using a 2N NaOH solution.
Preparation of negative control serum
Collecting blood serum of healthy blood donor without platelet antibody, adding sodium azide with final concentration of 0.1% after constant volume, and mixing uniformly
Positive control serum preparation
Screening out serum of healthy blood donor containing platelet antibody, adding sodium azide with final concentration of 0.1% after constant volume, and mixing uniformly.
Example 2 platelet Cross-matching
The kit was removed from the refrigerator and allowed to equilibrate to room temperature, and the strips were removed according to the number of tests.
Marking the drawn microplate strips, such as test wells, negative control wells, positive control wells, and the like.
Platelet suspensions from different donors, 100X 109/ml in count, 50uL per well, were added to each of the 6 wells from top to bottom, and the sides of the strips were tapped and mixed.
Placing the reaction plate strips into a plate centrifuge, centrifuging for 5 minutes at 50g, taking out the reaction plate strips, reversely and lightly throwing away unbound platelet suspension, washing for 3 times by using 1 XPBS (phosphate buffer solution), reversely and lightly throwing away washing liquid each time, and after the last light drying, reversely putting the reaction plate strips on water absorption paper, and sucking off residual liquid in holes.
Low ionic medium, 100 uL/well was added to the reaction wells, followed by patient serum or plasma, 50 uL/well, negative and positive control serum, 50 uL/well, respectively, in the negative and positive control wells.
The reaction plate was gently shaken at 900rpm for 10 seconds in a plate shaker or gently tapped by hand at the edge of the reaction plate to mix the well with the liquid.
Sealing the lath by the sealing film, placing the lath in a water bath at 37 ℃ for 30 minutes, or placing the lath in an air bath for 35 minutes, taking out the lath, tearing off the sealing film, reversely screwing off the liquid in the hole, washing the lath for 5 times by 1 XPBS (phosphate buffer solution), slightly drying the lath for the last time, reversely screwing the lath on absorbent paper, and sucking the residual liquid in the hole.
After 1 drop (50. Mu.L) of goat/rabbit anti-human IgG was added, 1 drop (50. Mu.L) of indicator red blood cells was immediately added, gently shaken and mixed well, and then placed in a plate centrifuge, and centrifuged at 200g for 5 minutes.
Taking out the lath and interpreting the result. The indicator cells are paved on the whole bottom of the reaction plate strip micropores, and the result is positive, which indicates that the cross-matching of the platelets is not suitable; if the indicating cells are gathered at the central bottom of the reaction plate strip micropore to form a cell buckle, judging the cell buckle to be negative, and indicating that the platelets are matched; if the morphology of the indicator cells at the bottom of the microplate well is between the two, the indicator cells are judged to be weak positive, indicating that the platelet cross-matching is not good (FIG. 2).
Example 3 kit stability test
The U-shaped reaction microplate prepared in example 1 was stored in a refrigerator at 2 to 8 ℃, and samples were taken for stability study at 2 months, 4 months, 6 months, 8 months, 10 months, and 12 months of the storage period, respectively; and reserving a patient sample with the platelet antibody, subpackaging and storing in a refrigerator at the temperature of-20 ℃. At each test, 1 piece of the test specimen was taken out of the refrigerator and thawed for use. The results show that the U-shaped reaction microplate prepared by the invention has no difference in cross matching use performance within 12 months of storage, and shows better stability, and the results are shown in figure 3.
1. The kit was removed from the refrigerator and allowed to equilibrate to room temperature, and the strips were removed therefrom according to the number of tests.
Marking the drawn microplate strips, such as test wells, negative control wells, positive control wells, and the like.
2. Platelet suspensions from different donors, 100X 109/ml in count, 50uL per well, were added to each of the 6 wells from top to bottom, and the sides of the strips were tapped and mixed.
3. Placing the reaction plate strips into a plate centrifuge, centrifuging for 5 minutes at 50g, taking out the reaction plate strips, reversely and lightly throwing away unbound platelet suspension, washing for 3 times by using 1 XPBS (phosphate buffer solution), reversely and lightly throwing away washing liquid each time, and after the last time of light spin-drying, reversely buckling the reaction plate strips on absorbent paper, and sucking off residual liquid in holes.
4. Low ionic medium, 100 uL/well, is added to the reaction wells, followed by patient serum or plasma, 50 uL/well, negative and positive control serum, 50 uL/well, respectively, in the negative and positive control wells.
5. The reaction plate was gently shaken at 900rpm for 10 seconds in a plate shaker or gently tapped by hand at the edge of the reaction plate to mix the well with the liquid.
6. Sealing the lath with a sealing film, placing in a water bath at 37 ℃ for 30 minutes, or placing in an air bath for 35 minutes, taking out, tearing off the sealing film, reversely screwing off the liquid in the hole, washing with 1 XPBS for 5 times, slightly spin-drying for the last time, reversely screwing the reaction lath on absorbent paper, and sucking off the residual liquid in the hole.
7. After 1 drop (50. Mu.L) of goat/rabbit anti-human IgG was added, 1 drop (50. Mu.L) of indicator red blood cells was immediately added, and after gently shaking and mixing, the mixture was centrifuged at 200g for 5 minutes in a plate centrifuge.
8. Taking out the lath and carrying out result interpretation. The indicating cells are paved on the whole bottom of the reaction plate strip micropores, and the result is positive, which indicates that the cross-matching of the platelets is not suitable; if the indicator cells are gathered at the central bottom of the reaction plate strip micropore to form a cell buckle, judging the cell buckle to be negative, and indicating that the platelet is matched and matched; if the morphology of the indicator cells at the bottom of the microplate well is between the two, the indicator cells are judged to be weak positive, indicating that the platelet cross-matching is not good (FIG. 3).
The above description is only a preferred embodiment of the present invention, the present invention is not limited to the above embodiment, and there may be some slight structural changes in the implementation, and if there are various changes or modifications to the present invention without departing from the spirit and scope of the present invention, and within the claims and equivalent technical scope of the present invention, the present invention is also intended to include those changes and modifications.
Claims (1)
1. A platelet cross-matching kit comprising:
(1) The preparation method comprises the following steps of (1) coating polyethyleneimine on the bottom of a hole of a U-shaped reaction microplate, and preparing the U-shaped reaction microplate by the following steps: preparing 0.1% polyethyleneimine solution by using 0.05M carbonate buffer solution with the pH value of 9.6, adding 50uL of the polyethyleneimine solution into a hole of a U-shaped reaction microplate with 96 holes per hole, placing the reaction microplate in a refrigerator with the temperature of 4 ℃ overnight, taking out the reaction microplate the next day, washing the reaction microplate for 3 times by using 1 XPBS buffer solution, throwing off the washing solution, finally, drying the residual liquid in the hole by using absorbent paper, bagging the reaction microplate by using aluminum foil, heating and sealing the reaction microplate, and placing the reaction microplate in the refrigerator with the temperature of 4 ℃ for later use; wherein, the U-shaped reaction microplate is made of a 96-hole U-shaped bottom detachable microplate strip;
(2) A low ionic medium which is a buffer comprising 1.8% glycine and 0.10% sodium azide;
(3) An indicator system comprising an IgG anti-D sensitized erythrocyte suspension and a rabbit or goat anti-human globulin reagent; wherein the sensitized erythrocyte suspension is prepared by adding IgG anti-D with the same volume into washed O-type packed erythrocytes, incubating for 50 minutes at 37 ℃ to prepare IgG anti-D sensitized erythrocytes, washing for 5 times by using normal saline, and preparing 0.35% suspension by using erythrocyte preservation solution, namely sensitized erythrocytes; anti-human globulin reagent: the rabbit or sheep anti-human globulin reagent is diluted by an antibody diluent in a gradient scale ratio, reacts with the sensitized red blood cells, is observed under a microscope after centrifugation to be a non-agglutination tube as the dilution factor of the anti-human globulin reagent, and the anti-human globulin reagent with the dilution factor and sensitized O-type red blood cells form an indicating system;
(4) Negative control serum, wherein the negative control serum is prepared by adding 0.1% of sodium azide into human serum which has no agglutination reaction with human platelets;
(5) And the positive control serum is obtained by adding 0.1% of sodium azide into human serum which has an agglutination reaction with human platelets.
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